We have already demonstrated that many cells in culture show gamma-glutamyltransferase (GammaGTF) activity in their cell membranes; and that, in particular, WI-38 and IMR-90 human fetal lung fibroblasts, diploid strains of finite life span, exhibit such activity throughout their life in culture. However, as we have shown, these cells in the stage of senescence preceding death of the cell culture, undergo a large increase in gammaGTF activity concomitant with very large increase in cell size. In that regard we have suggested that the change in cell size may alter the architecture of topology of the cell surface so that the gammaGTF is more available for reaction with the external substrates present in the medium. We have now purified the gammaGTF from WI-38 cells of intermediate levels of population doubling, partially characterized it and prepared antibody against the holoenzyme. We also have shown that the enzyme is inactivated by the fluorescent labeling reagent, 5-iodoacetamidofluorescein (5-IAF), and that the light subunit of the enzyme specifically takes up one mole equivalent of acetoamidofluorescein groups. We now propose to isolate the enzyme from both young and old cultures of IMR-90 fibroblasts, and determine whether there are age-related changes in Km values with different substrates, changes in isozyme patterns (if any), changes in heat stability, changes in amino acid composition of subunits, changes in immunologic properties and changes in binding of 5-IAF. Using whole cells of young and old cultures we shall study possible age-related alterations with respect to disposition of gammaGTF in the cell membranes as determined by reaction with 5-IAF and resultant localization of fluorescence in subunits. In young and old cells we shall also study changes in location of the enzyme in membranes by use of specific fluorescent antibodies directed against holoenzyme and against light and heavy subunits respectively. We shall also study another cell strain of finite life span, namely baby Cebus monkey kidney epithelial cells, since these have much higher intrinsic gammaGTF activity. These studies may have significance with respect to transport of amino acids and peptides in old as opposed to young cells and tissues.